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MedChemExpress tg2 in 1
Tg2 In 1, supplied by MedChemExpress, used in various techniques. Bioz Stars score: 93/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/tg2+in+1/pmc13040212-349-10-12?v=MedChemExpress
Average 93 stars, based on 3 article reviews

tg2 in 1 - by Bioz Stars, 2026-07

93/100 stars

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Effects of 1-155 on viability and FN deposition by pHPFs. ( A ) pHPFs (5000 cells/well in 96-well plates) were treated with 1-155 at 10 µM and 25 µM, while pulmonary cell culture medium and DMSO (1:1000 dilution) were used as control treatments. Cell viability was measured using XTT assay at 24, 48, and 72 h. Absorbance at 490 nm and 630 nm was measured using plate reader. n = 5. ( B ) Structure of 1-155. ( C ) FN matrix deposition by human pulmonary fibroblasts were detected via immunofluorescence staining as described in Materials and Methods. pHPFs were treated with 1 ng/mL of TGFβ1 in presence of TG2 inhibitor 1-155 at concentrations between 50 and 500 nM, while DMSO (1:1000 dilution) was used as vehicle control. Mouse anti-human FN primary antibody and GFP-tagged secondary antibody were used and fluorescence signal was measured using EPI-fluorescence microscope. ImageJ was used to measure fluorescence signal (n = 3). Bar, 25 µm.

Journal: Cells

Article Title: Inhibition of Transglutaminase 2 by a Selective Small Molecule Inhibitor Reduces Fibrosis and Improves Pulmonary Function in a Bleomycin Mouse Model

doi: 10.3390/cells14070497

Figure Lengend Snippet: Effects of 1-155 on viability and FN deposition by pHPFs. ( A ) pHPFs (5000 cells/well in 96-well plates) were treated with 1-155 at 10 µM and 25 µM, while pulmonary cell culture medium and DMSO (1:1000 dilution) were used as control treatments. Cell viability was measured using XTT assay at 24, 48, and 72 h. Absorbance at 490 nm and 630 nm was measured using plate reader. n = 5. ( B ) Structure of 1-155. ( C ) FN matrix deposition by human pulmonary fibroblasts were detected via immunofluorescence staining as described in Materials and Methods. pHPFs were treated with 1 ng/mL of TGFβ1 in presence of TG2 inhibitor 1-155 at concentrations between 50 and 500 nM, while DMSO (1:1000 dilution) was used as vehicle control. Mouse anti-human FN primary antibody and GFP-tagged secondary antibody were used and fluorescence signal was measured using EPI-fluorescence microscope. ImageJ was used to measure fluorescence signal (n = 3). Bar, 25 µm.

Article Snippet: TG2 inhibitor 1-155 (with >95% purity) was synthesized by Wuxi AppTec (Shanghai, China).

Techniques: Cell Culture, Control, XTT Assay, Immunofluorescence, Staining, Fluorescence, Microscopy

The effects of 1-155 on the presence of TG2 and αSMA antigen in the lung tissues in the bleomycin mouse model. As described in Materials and Methods, a rabbit anti-TG2 polyclonal antibody and a rabbit anti- α-SMA polyclonal antibody were used to detect TG2 ( A , B ) and α-SMA ( C , D ) in the lung tissues in the bleomycin-induced pulmonary fibrosis mouse model via immunohistochemistry. The samples from the saline control, 1-155 IN, and its vehicle control treatments were analyzed. Representative images of TG2 ( A ) and α-SMA staining ( C ) are shown. ImageJ was used for data analysis, as shown in ( B , D ). *, p < 0.05; ***, p < 0.0001. n = 6. Bar, 100 µm.

Journal: Cells

Article Title: Inhibition of Transglutaminase 2 by a Selective Small Molecule Inhibitor Reduces Fibrosis and Improves Pulmonary Function in a Bleomycin Mouse Model

doi: 10.3390/cells14070497

Figure Lengend Snippet: The effects of 1-155 on the presence of TG2 and αSMA antigen in the lung tissues in the bleomycin mouse model. As described in Materials and Methods, a rabbit anti-TG2 polyclonal antibody and a rabbit anti- α-SMA polyclonal antibody were used to detect TG2 ( A , B ) and α-SMA ( C , D ) in the lung tissues in the bleomycin-induced pulmonary fibrosis mouse model via immunohistochemistry. The samples from the saline control, 1-155 IN, and its vehicle control treatments were analyzed. Representative images of TG2 ( A ) and α-SMA staining ( C ) are shown. ImageJ was used for data analysis, as shown in ( B , D ). *, p < 0.05; ***, p < 0.0001. n = 6. Bar, 100 µm.

Article Snippet: TG2 inhibitor 1-155 (with >95% purity) was synthesized by Wuxi AppTec (Shanghai, China).

Techniques: Immunohistochemistry, Saline, Control, Staining

Tgm2 is highly expressed in senescent microglia. (A) Representative immunohistochemical images of Tgm2 protein in the hippocampal tissue of young (4‐month) and old (22‐month) C57/B6 mice. (B, C) The protein abundance relative expression level of Tgm2 in the hippocampus of C57/B6 mice by western blotting analysis ( n = 3). (D) A workflow for isolating and identifying senescent primary microglia from 22‐month‐old mice ( n = 3). (E) Western blot analysis of Tgm2 and P21 protein levels in primary microglia. (F, G) The protein abundance relative expression level of Tgm2 and P21 by western blot analysis ( n = 3). Young, Young mice or young cells; Sene, Senescent cells. Data were analyzed by Student's t ‐test. p > 0.05 is indicated by ns; p < 0.05 is indicated by ⁎; p < 0.01 is indicated by ⁎⁎; p < 0.0001 are indicated by ⁎⁎⁎⁎.

Journal: Aging Cell

Article Title: Tgm2‐Catalyzed Covalent Cross‐Linking of IκBα Drives NF ‐ κB Nuclear Translocation to Promote SASP in Senescent Microglia

doi: 10.1111/acel.14463

Figure Lengend Snippet: Tgm2 is highly expressed in senescent microglia. (A) Representative immunohistochemical images of Tgm2 protein in the hippocampal tissue of young (4‐month) and old (22‐month) C57/B6 mice. (B, C) The protein abundance relative expression level of Tgm2 in the hippocampus of C57/B6 mice by western blotting analysis ( n = 3). (D) A workflow for isolating and identifying senescent primary microglia from 22‐month‐old mice ( n = 3). (E) Western blot analysis of Tgm2 and P21 protein levels in primary microglia. (F, G) The protein abundance relative expression level of Tgm2 and P21 by western blot analysis ( n = 3). Young, Young mice or young cells; Sene, Senescent cells. Data were analyzed by Student's t ‐test. p > 0.05 is indicated by ns; p < 0.05 is indicated by ⁎; p < 0.01 is indicated by ⁎⁎; p < 0.0001 are indicated by ⁎⁎⁎⁎.

Article Snippet: Cys‐D (Cat# A53299, China) was purchased from OKA, Tg2‐IN1 (Cat# HY117678 , USA) was purchased from MCE, and etoposide (Cat# SC0173, China) was purchased from Beyotime.

Techniques: Immunohistochemical staining, Quantitative Proteomics, Expressing, Western Blot

Tgm2 is highly expressed in senescent BV2 cells. (A) Western blotting analysis of Tgm2 in senescent BV2 cells. (B) Graphs represent the quantification of the Tgm2 blots ( n = 3). (C) Immunofluorescence images of Tgm2 protein in control and senescent BV2 cells. Scale bar: 100 μm. (D) The TMT quantitative proteomics analysis workflow for senescent BV2 cells. (E, F) The heatmap and volcano plot depicted the difference in protein levels between control cells (Ctrl) and senescent cells (Sene). (G) The relative protein abundance of Tgm2 in BV2 cells was determined by TMT quantitative proteomics analysis ( n = 3). Ctrl, control cells; Sene, senescent cells. Student's t ‐test analyzed data. p < 0.0001 are indicated by ⁎⁎⁎⁎.

Journal: Aging Cell

Article Title: Tgm2‐Catalyzed Covalent Cross‐Linking of IκBα Drives NF ‐ κB Nuclear Translocation to Promote SASP in Senescent Microglia

doi: 10.1111/acel.14463

Figure Lengend Snippet: Tgm2 is highly expressed in senescent BV2 cells. (A) Western blotting analysis of Tgm2 in senescent BV2 cells. (B) Graphs represent the quantification of the Tgm2 blots ( n = 3). (C) Immunofluorescence images of Tgm2 protein in control and senescent BV2 cells. Scale bar: 100 μm. (D) The TMT quantitative proteomics analysis workflow for senescent BV2 cells. (E, F) The heatmap and volcano plot depicted the difference in protein levels between control cells (Ctrl) and senescent cells (Sene). (G) The relative protein abundance of Tgm2 in BV2 cells was determined by TMT quantitative proteomics analysis ( n = 3). Ctrl, control cells; Sene, senescent cells. Student's t ‐test analyzed data. p < 0.0001 are indicated by ⁎⁎⁎⁎.

Article Snippet: Cys‐D (Cat# A53299, China) was purchased from OKA, Tg2‐IN1 (Cat# HY117678 , USA) was purchased from MCE, and etoposide (Cat# SC0173, China) was purchased from Beyotime.

Techniques: Western Blot, Immunofluorescence, Control, Quantitative Proteomics

The NF‐κB pathway is activated in senescent BV2 cells and the brain tissues of aged mice. (A) Western blotting analysis of Tgm2, P105, p‐P65, and IκBα protein levels in senescent BV2 cells ( n = 3). (B) Western blotting analysis of Tgm2, P105, p‐P65, and IκBα protein levels in the olfactory bulb, cortex, and hippocampus tissues of C57/B6 mice ( n = 3). Data were analyzed by Student's t ‐test. p > 0.05 is indicated by ns; p < 0.05 is indicated by ⁎; p < 0.01 is indicated by ⁎⁎; p < 0.001 is indicated by ⁎⁎⁎; p < 0.0001 is indicated by ⁎⁎⁎⁎.

Journal: Aging Cell

Article Title: Tgm2‐Catalyzed Covalent Cross‐Linking of IκBα Drives NF ‐ κB Nuclear Translocation to Promote SASP in Senescent Microglia

doi: 10.1111/acel.14463

Figure Lengend Snippet: The NF‐κB pathway is activated in senescent BV2 cells and the brain tissues of aged mice. (A) Western blotting analysis of Tgm2, P105, p‐P65, and IκBα protein levels in senescent BV2 cells ( n = 3). (B) Western blotting analysis of Tgm2, P105, p‐P65, and IκBα protein levels in the olfactory bulb, cortex, and hippocampus tissues of C57/B6 mice ( n = 3). Data were analyzed by Student's t ‐test. p > 0.05 is indicated by ns; p < 0.05 is indicated by ⁎; p < 0.01 is indicated by ⁎⁎; p < 0.001 is indicated by ⁎⁎⁎; p < 0.0001 is indicated by ⁎⁎⁎⁎.

Article Snippet: Cys‐D (Cat# A53299, China) was purchased from OKA, Tg2‐IN1 (Cat# HY117678 , USA) was purchased from MCE, and etoposide (Cat# SC0173, China) was purchased from Beyotime.

Techniques: Western Blot

Tgm2 inhibitors (CD and TIN1) treatment reduces the NF‐κB nuclear translocation in senescent BV2 cells. (A) Western blotting analysis of Tgm2, P65, p‐P65, P50, p‐IκBα, and IκBα protein levels in cytosol of BV2 cells ( n = 3). (B) Western blotting analysis of Tgm2, P65, p‐P65, P50, p‐P50, and IκBα protein levels in nucleus of BV2 cells ( n = 3). (C) Tgm2 inhibitor treatment reduces the P65 in the nucleus of senescent BV2 cells by immunofluorescence. TIN1: TG‐2‐IN‐1 (CAS No.: 135273‐74‐4) is a Tgm2 inhibitor. ETOP + TIN1: BV2 cells were treated with 10 μM etoposide and 200 μM TIN1 together. Scale bar: 10 μm. Data were analyzed by one‐way ANOVA. p > 0.05 is indicated by ns; p < 0.05 is indicated by ⁎; p < 0.01 is indicated by ⁎⁎; p < 0.001 is indicated by ⁎⁎⁎; p < 0.0001 is indicated by ⁎⁎⁎⁎.

Journal: Aging Cell

Article Title: Tgm2‐Catalyzed Covalent Cross‐Linking of IκBα Drives NF ‐ κB Nuclear Translocation to Promote SASP in Senescent Microglia

doi: 10.1111/acel.14463

Figure Lengend Snippet: Tgm2 inhibitors (CD and TIN1) treatment reduces the NF‐κB nuclear translocation in senescent BV2 cells. (A) Western blotting analysis of Tgm2, P65, p‐P65, P50, p‐IκBα, and IκBα protein levels in cytosol of BV2 cells ( n = 3). (B) Western blotting analysis of Tgm2, P65, p‐P65, P50, p‐P50, and IκBα protein levels in nucleus of BV2 cells ( n = 3). (C) Tgm2 inhibitor treatment reduces the P65 in the nucleus of senescent BV2 cells by immunofluorescence. TIN1: TG‐2‐IN‐1 (CAS No.: 135273‐74‐4) is a Tgm2 inhibitor. ETOP + TIN1: BV2 cells were treated with 10 μM etoposide and 200 μM TIN1 together. Scale bar: 10 μm. Data were analyzed by one‐way ANOVA. p > 0.05 is indicated by ns; p < 0.05 is indicated by ⁎; p < 0.01 is indicated by ⁎⁎; p < 0.001 is indicated by ⁎⁎⁎; p < 0.0001 is indicated by ⁎⁎⁎⁎.

Article Snippet: Cys‐D (Cat# A53299, China) was purchased from OKA, Tg2‐IN1 (Cat# HY117678 , USA) was purchased from MCE, and etoposide (Cat# SC0173, China) was purchased from Beyotime.

Techniques: Translocation Assay, Western Blot, Immunofluorescence

CD treatment reduces SASP factors in senescent BV2 cells. (A) SA‐β‐Gal staining results of BV2 cells. Ctrl, control cells; Sene, Senescent cells. Sene+CD: BV2 cells were treated with 10 μM etoposide and 500 μM CD together. Scale bar: 50 μm. (B, C) RT‐qPCR was used to measure mRNA levels of IL6 , MMP3 , MMP12 , CXCL1 , CXCL2 , CXCL10 , P21 , and P53 ( n = 3). Ctrl, control cells. CD: BV2 cells were treated with 500 μM CD. ETOP: BV2 cells were treated with 10 μM etoposide to induce DNA damage‐induced senescence. ETOP+CD: BV2 cells were treated with 10 μM etoposide and 500 μM CD together. (D) RT‐qPCR was used to measure mRNA levels of IL6 , MMP3 , and CXCL1 ( n = 4). NC, negative control BV2 cells. NC + ETOP: Negative control BV2 cells were treated with 10 μM etoposide. TKD + ETOP: BV2 cells with Tgm2 knockdown were treated with 10 μM etoposide. Data were analyzed by one‐way ANOVA or Student's t ‐test. p > 0.05 is indicated by ns; p < 0.05 is indicated by ⁎; p < 0.01 is indicated by ⁎⁎; p < 0.001 is indicated by ⁎⁎⁎; p < 0.0001 is indicated by ⁎⁎⁎⁎.

Journal: Aging Cell

Article Title: Tgm2‐Catalyzed Covalent Cross‐Linking of IκBα Drives NF ‐ κB Nuclear Translocation to Promote SASP in Senescent Microglia

doi: 10.1111/acel.14463

Figure Lengend Snippet: CD treatment reduces SASP factors in senescent BV2 cells. (A) SA‐β‐Gal staining results of BV2 cells. Ctrl, control cells; Sene, Senescent cells. Sene+CD: BV2 cells were treated with 10 μM etoposide and 500 μM CD together. Scale bar: 50 μm. (B, C) RT‐qPCR was used to measure mRNA levels of IL6 , MMP3 , MMP12 , CXCL1 , CXCL2 , CXCL10 , P21 , and P53 ( n = 3). Ctrl, control cells. CD: BV2 cells were treated with 500 μM CD. ETOP: BV2 cells were treated with 10 μM etoposide to induce DNA damage‐induced senescence. ETOP+CD: BV2 cells were treated with 10 μM etoposide and 500 μM CD together. (D) RT‐qPCR was used to measure mRNA levels of IL6 , MMP3 , and CXCL1 ( n = 4). NC, negative control BV2 cells. NC + ETOP: Negative control BV2 cells were treated with 10 μM etoposide. TKD + ETOP: BV2 cells with Tgm2 knockdown were treated with 10 μM etoposide. Data were analyzed by one‐way ANOVA or Student's t ‐test. p > 0.05 is indicated by ns; p < 0.05 is indicated by ⁎; p < 0.01 is indicated by ⁎⁎; p < 0.001 is indicated by ⁎⁎⁎; p < 0.0001 is indicated by ⁎⁎⁎⁎.

Article Snippet: Cys‐D (Cat# A53299, China) was purchased from OKA, Tg2‐IN1 (Cat# HY117678 , USA) was purchased from MCE, and etoposide (Cat# SC0173, China) was purchased from Beyotime.

Techniques: Staining, Control, Quantitative RT-PCR, Negative Control, Knockdown

Tgm2 catalyzes the covalent cross‐linking of the residues K22 and Q248 of IκBa in the cytoplasm of BV2 cells, resulting in the dimerization of IκBa. (A) The nondenaturing western blot result for IκBα protein in the cytosol of senescent BV2 cells. (B) The nondenaturing western blot result demonstrates that Tgm2 protein promotes the cross‐linking of IκBα into dimers in the cytosol of senescent BV2 cells in vitro. (C) The IκBa dimer structure, predicted by AlphaFold 3.0, shows the two molecules are centrosymmetric. (D) The MS2 spectra of the Tgm2–cross‐linked IκBα peptide segments in the cytosol of senescent BV2 cells. Data were analyzed by Student's t ‐test. p < 0.05 is indicated by ⁎.

Journal: Aging Cell

Article Title: Tgm2‐Catalyzed Covalent Cross‐Linking of IκBα Drives NF ‐ κB Nuclear Translocation to Promote SASP in Senescent Microglia

doi: 10.1111/acel.14463

Figure Lengend Snippet: Tgm2 catalyzes the covalent cross‐linking of the residues K22 and Q248 of IκBa in the cytoplasm of BV2 cells, resulting in the dimerization of IκBa. (A) The nondenaturing western blot result for IκBα protein in the cytosol of senescent BV2 cells. (B) The nondenaturing western blot result demonstrates that Tgm2 protein promotes the cross‐linking of IκBα into dimers in the cytosol of senescent BV2 cells in vitro. (C) The IκBa dimer structure, predicted by AlphaFold 3.0, shows the two molecules are centrosymmetric. (D) The MS2 spectra of the Tgm2–cross‐linked IκBα peptide segments in the cytosol of senescent BV2 cells. Data were analyzed by Student's t ‐test. p < 0.05 is indicated by ⁎.

Article Snippet: Cys‐D (Cat# A53299, China) was purchased from OKA, Tg2‐IN1 (Cat# HY117678 , USA) was purchased from MCE, and etoposide (Cat# SC0173, China) was purchased from Beyotime.

Techniques: Western Blot, In Vitro

CD (Tgm2 enzyme inhibitor) can alleviate the senescent phenotype of aged mice. (A) The timeline of the experiment using CD to treat aged mice. (B) The trend of weight change in aged mice. (C) CD improves hair loss in aged mice. C1: Mouse were gavaged with H 2 O; C2: Mouse were gavaged with CD ( n = 8). (D) The results of the rotarod test in aged mice after CD treatment. (E) The results of the open‐field test in aged mice after CD treatment. (F) The results of the grip strength test in aged mice after CD treatment. (G) The results of the Y‐maze test in aged mice after CD treatment. (H) CD reduces the levels of senescence and inflammation markers in the hippocampus. Data were analyzed by Student's t ‐test. p > 0.05 is indicated by ns; p < 0.05 is indicated by ⁎; p < 0.01 is indicated by ⁎⁎; p < 0.001 is indicated by ⁎⁎⁎.

Journal: Aging Cell

Article Title: Tgm2‐Catalyzed Covalent Cross‐Linking of IκBα Drives NF ‐ κB Nuclear Translocation to Promote SASP in Senescent Microglia

doi: 10.1111/acel.14463

Figure Lengend Snippet: CD (Tgm2 enzyme inhibitor) can alleviate the senescent phenotype of aged mice. (A) The timeline of the experiment using CD to treat aged mice. (B) The trend of weight change in aged mice. (C) CD improves hair loss in aged mice. C1: Mouse were gavaged with H 2 O; C2: Mouse were gavaged with CD ( n = 8). (D) The results of the rotarod test in aged mice after CD treatment. (E) The results of the open‐field test in aged mice after CD treatment. (F) The results of the grip strength test in aged mice after CD treatment. (G) The results of the Y‐maze test in aged mice after CD treatment. (H) CD reduces the levels of senescence and inflammation markers in the hippocampus. Data were analyzed by Student's t ‐test. p > 0.05 is indicated by ns; p < 0.05 is indicated by ⁎; p < 0.01 is indicated by ⁎⁎; p < 0.001 is indicated by ⁎⁎⁎.

Article Snippet: Cys‐D (Cat# A53299, China) was purchased from OKA, Tg2‐IN1 (Cat# HY117678 , USA) was purchased from MCE, and etoposide (Cat# SC0173, China) was purchased from Beyotime.

Techniques:

A schematic summary of the Tgm2‐NF‐κB‐SASP loop pathway in senescent microglia.

Journal: Aging Cell

Article Title: Tgm2‐Catalyzed Covalent Cross‐Linking of IκBα Drives NF ‐ κB Nuclear Translocation to Promote SASP in Senescent Microglia

doi: 10.1111/acel.14463

Figure Lengend Snippet: A schematic summary of the Tgm2‐NF‐κB‐SASP loop pathway in senescent microglia.

Article Snippet: Cys‐D (Cat# A53299, China) was purchased from OKA, Tg2‐IN1 (Cat# HY117678 , USA) was purchased from MCE, and etoposide (Cat# SC0173, China) was purchased from Beyotime.

Techniques:

(A) Tet-on vector, Tet-on wtTG2 and Tet-on TG2C277S cells were treated with 50 µM doxycycline (Dox). The expression level of TG2 protein was detected by Western blot analysis after 18 h of Dox treatment. β-actin was used as a loading control. (B) The transglutaminase enzyme activity in the various types of Jurkat cells was determined after 6 h of Dox treatment. (C) Induced overexpression of wtTG2 or TG2C277S decreased the cell viability in a time dependent manner. Number of viable cells was determined at the indicated time points following Dox treatment. Significantly different from the viable cell number of the indicated cell line at the same time point (*** P <0.001). (D) Induced overexpression of wtTG2 or TG2C277S increased the percentage of Annexin V positive cells. Number of Annexin V and/or propidium iodide positive cells was determined at the indicated time points following Dox treatment. Significantly different from the % of Annexin V positive cells of the indicated cell line at the same time point (** P <0.01; *** P <0.001). Flow cytometric data demonstrate results detected after 48 h of Dox treatment. (E) DNA histogram of propidium iodide stained Tet-on vector, Tet-on wtTG2 and Tet-on TG2C277S cells treated with Dox for 60 hours. Cells exhibiting sub-G1 levels of DNA were considered apoptotic and their amount was calculated as percentage of the total cell number. *Data represent at least three independent experiments and are shown as mean ± SD ( P <0.05). (F) Electrophoretic analysis of internucleosomal DNA fragmentation in Tet-on vector, Tet-on wtTG2 and Tet-on TG2C277S cells treated with or without Dox (50 µM) for 60 hours. MW, standard. The results are representative of one of three independent experiments.

Journal: PLoS ONE

Article Title: Transglutaminase 2 Contributes to Apoptosis Induction in Jurkat T Cells by Modulating Ca 2+ Homeostasis via Cross-Linking RAP1GDS1

doi: 10.1371/journal.pone.0081516

Figure Lengend Snippet: (A) Tet-on vector, Tet-on wtTG2 and Tet-on TG2C277S cells were treated with 50 µM doxycycline (Dox). The expression level of TG2 protein was detected by Western blot analysis after 18 h of Dox treatment. β-actin was used as a loading control. (B) The transglutaminase enzyme activity in the various types of Jurkat cells was determined after 6 h of Dox treatment. (C) Induced overexpression of wtTG2 or TG2C277S decreased the cell viability in a time dependent manner. Number of viable cells was determined at the indicated time points following Dox treatment. Significantly different from the viable cell number of the indicated cell line at the same time point (*** P <0.001). (D) Induced overexpression of wtTG2 or TG2C277S increased the percentage of Annexin V positive cells. Number of Annexin V and/or propidium iodide positive cells was determined at the indicated time points following Dox treatment. Significantly different from the % of Annexin V positive cells of the indicated cell line at the same time point (** P <0.01; *** P <0.001). Flow cytometric data demonstrate results detected after 48 h of Dox treatment. (E) DNA histogram of propidium iodide stained Tet-on vector, Tet-on wtTG2 and Tet-on TG2C277S cells treated with Dox for 60 hours. Cells exhibiting sub-G1 levels of DNA were considered apoptotic and their amount was calculated as percentage of the total cell number. *Data represent at least three independent experiments and are shown as mean ± SD ( P <0.05). (F) Electrophoretic analysis of internucleosomal DNA fragmentation in Tet-on vector, Tet-on wtTG2 and Tet-on TG2C277S cells treated with or without Dox (50 µM) for 60 hours. MW, standard. The results are representative of one of three independent experiments.

Article Snippet: Antibodies were purchased from the following sources and used at the indicated dilutions: RAP1GDS1 (1∶500) from Santa Cruz, TG2 (1∶500) from Thermo Scientific, anti-ε(γ-glutamyl) lysine isopeptide (1∶500) from Covalab, β–tubulin (1∶3000), Hsp60 (1∶1000) and Sigma-1R (1∶1000) from Sigma-Aldrich; IP3R3 (1∶300) from BD Biosciences; VDAC (1∶5000) from Abcam; β-actin (1∶1000) from Merck Millipore.

Techniques: Plasmid Preparation, Expressing, Western Blot, Activity Assay, Over Expression, Staining

(A) Time dependent changes in the mitochondrial Ca 2+ concentrations of Tet-on vector, Tet-on wtTG2 and Tet-on TG2C277S cells following Dox (50 µM) treatment detected by Rhod-2/AM. (B) Time dependent changes in the cytosolic Ca 2+ concentrations of Tet-on vector, Tet-on wtTG2 and Tet-on TG2C277S cells following Dox (50 µM) treatment detected by Fura-2/AM. (C) Time dependent changes in the mitochondrial and cytoplasmic TG2 expressions of Tet-on wtTG2 and Tet-on TG2C277S cells following Dox (50 µM) treatment detected by Western blot analysis. Mitochondrial HSP60 and cytoplasmic β-actin were used as loading controls; while β-tubulin was used to check for cytoplasmic contamination of mitochondria. (D) Time dependent changes in the mitochondrial and cytoplasmic TG2 activities of Tet-on vector, Tet-on wtTG2 and Tet-on TG2C277S cells following Dox (50 µM) treatment. (E) Confocal images taken at 16 h following Dox treatment show Tet-on wtTG2 cells expressing TG2 (green) colocalized with the mitochondrial calcium indicator Rhod-2/AM (red). Scale bar = 5 µM. All the data presented represent mean±S.D. of at least three determinations. Significantly different from the TG2C277S cell line detected at the same time point (* P <0.5; ** P <0.01; *** P <0.001).

Journal: PLoS ONE

Article Title: Transglutaminase 2 Contributes to Apoptosis Induction in Jurkat T Cells by Modulating Ca 2+ Homeostasis via Cross-Linking RAP1GDS1

doi: 10.1371/journal.pone.0081516

Figure Lengend Snippet: (A) Time dependent changes in the mitochondrial Ca 2+ concentrations of Tet-on vector, Tet-on wtTG2 and Tet-on TG2C277S cells following Dox (50 µM) treatment detected by Rhod-2/AM. (B) Time dependent changes in the cytosolic Ca 2+ concentrations of Tet-on vector, Tet-on wtTG2 and Tet-on TG2C277S cells following Dox (50 µM) treatment detected by Fura-2/AM. (C) Time dependent changes in the mitochondrial and cytoplasmic TG2 expressions of Tet-on wtTG2 and Tet-on TG2C277S cells following Dox (50 µM) treatment detected by Western blot analysis. Mitochondrial HSP60 and cytoplasmic β-actin were used as loading controls; while β-tubulin was used to check for cytoplasmic contamination of mitochondria. (D) Time dependent changes in the mitochondrial and cytoplasmic TG2 activities of Tet-on vector, Tet-on wtTG2 and Tet-on TG2C277S cells following Dox (50 µM) treatment. (E) Confocal images taken at 16 h following Dox treatment show Tet-on wtTG2 cells expressing TG2 (green) colocalized with the mitochondrial calcium indicator Rhod-2/AM (red). Scale bar = 5 µM. All the data presented represent mean±S.D. of at least three determinations. Significantly different from the TG2C277S cell line detected at the same time point (* P <0.5; ** P <0.01; *** P <0.001).

Techniques: Plasmid Preparation, Western Blot, Expressing

Tet-on vector, Tet-on wtTG2 and Tet-on TG2C277S cells treated with Dox (50 µM) for 18 h were exposed to 5 µM thapsigargin (tg), and changes in the Ca 2+ concentrations in the mitochondria (A) or in the ER (B) were monitored as described in the Materials and Methods. Wild type and TG2KO MEF cells were exposed to 5 µM thapsigarginin Ca 2+ free medium and changes in the Ca 2+ concentrations in the mitochondria (C) or in the ER (D) were monitored as described in the Materials and Methods. ( E ) Wild type and TG2 KO MEF cells were exposed to 500 µM ATP in Ca 2+ -free medium, and changes in the intra-mitochondrial Ca 2+ concentrations were monitored as described in the Materials and Methods. Left panels , Representative kinetic average changes in mitochondrial or ER Ca 2+ signals induced by thapsigarginor ATP over time are shown. Right panels , Areas, statistical evaluation of integrated Ca 2+ responses are shown. AUC, area under the curve. These data are representative of at least three experiments and shown as mean ± SD. * P <0.05; ***, P <0.001.

Journal: PLoS ONE

Article Title: Transglutaminase 2 Contributes to Apoptosis Induction in Jurkat T Cells by Modulating Ca 2+ Homeostasis via Cross-Linking RAP1GDS1

doi: 10.1371/journal.pone.0081516

Figure Lengend Snippet: Tet-on vector, Tet-on wtTG2 and Tet-on TG2C277S cells treated with Dox (50 µM) for 18 h were exposed to 5 µM thapsigargin (tg), and changes in the Ca 2+ concentrations in the mitochondria (A) or in the ER (B) were monitored as described in the Materials and Methods. Wild type and TG2KO MEF cells were exposed to 5 µM thapsigarginin Ca 2+ free medium and changes in the Ca 2+ concentrations in the mitochondria (C) or in the ER (D) were monitored as described in the Materials and Methods. ( E ) Wild type and TG2 KO MEF cells were exposed to 500 µM ATP in Ca 2+ -free medium, and changes in the intra-mitochondrial Ca 2+ concentrations were monitored as described in the Materials and Methods. Left panels , Representative kinetic average changes in mitochondrial or ER Ca 2+ signals induced by thapsigarginor ATP over time are shown. Right panels , Areas, statistical evaluation of integrated Ca 2+ responses are shown. AUC, area under the curve. These data are representative of at least three experiments and shown as mean ± SD. * P <0.05; ***, P <0.001.

Techniques: Plasmid Preparation

(A–C) Representative recordings of thapsigargin-induced intra-mitochondrial Ca 2+ elevations in Tet-on cells treated previously with Dox (50 µM) for 18 h, in the presence or absence of 1 µM TMB-8, an Ins 3 P receptor antagonist, 1 µM ryanodine, or both. Tg response of Tet-on vector and Tet-on TG2C277S cells treated with Dox (50 µM) for 18 h is also shown. (D–F) Representative recordings of tg-induced intra-mitochondrial Ca 2+ elevations in in WT MEF and TG2 KO MEF cells in the presence or absence of 1 µM TMB-8, 1 µM Ryanodine or both. Top panels , Representative kinetic average changes in mitochondrial Ca 2+ signals induced by tg (5 µM) over time are shown. Bottom panels , Areas, statistical evaluation of integrated Ca 2+ responses are shown. AUC, area under the curve. (G) Resting value of Mag-Fura 2 ratios (340/380) were measured in Mag-fura-2/AM loaded WT MEF and TG2 KO MEF cells. All the presented data are representative of at least three experiments and are shown as mean ± SD. *, P <0.05; ***, P <0.00; n.s., no significance.

Journal: PLoS ONE

Article Title: Transglutaminase 2 Contributes to Apoptosis Induction in Jurkat T Cells by Modulating Ca 2+ Homeostasis via Cross-Linking RAP1GDS1

doi: 10.1371/journal.pone.0081516

Figure Lengend Snippet: (A–C) Representative recordings of thapsigargin-induced intra-mitochondrial Ca 2+ elevations in Tet-on cells treated previously with Dox (50 µM) for 18 h, in the presence or absence of 1 µM TMB-8, an Ins 3 P receptor antagonist, 1 µM ryanodine, or both. Tg response of Tet-on vector and Tet-on TG2C277S cells treated with Dox (50 µM) for 18 h is also shown. (D–F) Representative recordings of tg-induced intra-mitochondrial Ca 2+ elevations in in WT MEF and TG2 KO MEF cells in the presence or absence of 1 µM TMB-8, 1 µM Ryanodine or both. Top panels , Representative kinetic average changes in mitochondrial Ca 2+ signals induced by tg (5 µM) over time are shown. Bottom panels , Areas, statistical evaluation of integrated Ca 2+ responses are shown. AUC, area under the curve. (G) Resting value of Mag-Fura 2 ratios (340/380) were measured in Mag-fura-2/AM loaded WT MEF and TG2 KO MEF cells. All the presented data are representative of at least three experiments and are shown as mean ± SD. *, P <0.05; ***, P <0.00; n.s., no significance.

Techniques: Plasmid Preparation

Expression of RAP1GDS1 analyzed by Western blotting in Tet-on wtTG2 and Tet-on TG2C277S cells treated or not with Dox (50 µM) for (A) 6 or (B) 18 hours. (C) Increasing amounts of cross-linked RAP1GDS1 in Tet-on wtTG2 cells treated with tg (5 µM) for 15 min, Dox for 6 hours and subsequently exposed to tg (5 µM) for 15 min analyzed by Western blotting. (D) Increasing amounts of cross-linked RAP1GDS1 in WT but not in TG2 KO MEF cells exposed to increasing concentrations of tg for 15 min analyzed by Western blotting. In all these experiments β-actin was used as loading control. (E) Tet-on wtTG2 cells were treated either with tg (5 µM) for 15 min, or with Dox for 6 hours. RAP1GDS1 was immunoprecipitated from the cell lysate and probed with anti-ε(γ-glutamyl) lysine isopeptide antibodies.

Journal: PLoS ONE

Article Title: Transglutaminase 2 Contributes to Apoptosis Induction in Jurkat T Cells by Modulating Ca 2+ Homeostasis via Cross-Linking RAP1GDS1

doi: 10.1371/journal.pone.0081516

Figure Lengend Snippet: Expression of RAP1GDS1 analyzed by Western blotting in Tet-on wtTG2 and Tet-on TG2C277S cells treated or not with Dox (50 µM) for (A) 6 or (B) 18 hours. (C) Increasing amounts of cross-linked RAP1GDS1 in Tet-on wtTG2 cells treated with tg (5 µM) for 15 min, Dox for 6 hours and subsequently exposed to tg (5 µM) for 15 min analyzed by Western blotting. (D) Increasing amounts of cross-linked RAP1GDS1 in WT but not in TG2 KO MEF cells exposed to increasing concentrations of tg for 15 min analyzed by Western blotting. In all these experiments β-actin was used as loading control. (E) Tet-on wtTG2 cells were treated either with tg (5 µM) for 15 min, or with Dox for 6 hours. RAP1GDS1 was immunoprecipitated from the cell lysate and probed with anti-ε(γ-glutamyl) lysine isopeptide antibodies.

Techniques: Expressing, Western Blot, Immunoprecipitation

(A) RAP1GDS1 and TG2 protein expression levels detected by Western blotting in Tet-on wtTG2, Tet-on wtTG2 with sh vector and Tet-on wtTG2 with shRAP1GDS1 (sh1) cells following 18 hours Dox (50 µM) treatment. β-actin was used as loading control. Tet-On wtTG2 with sh vector and sh1 cells treated with Dox for 18 hours were exposed to 5 µM thapsigargin. (B) A representative recording of the tg-induced Ca 2+ release from the ER recorded by Mag-Fura-2/AM fluorescence is shown. Right panel , statistical evaluation of the tg-induced ER Ca 2+ depletion. (C) A representative recording of the tg-induced intra-mitochondrial Ca 2+ changes is shown. Right panel , Area, statistical evaluation of integrated Ca 2+ response. AUC, area under the curve. These data are representative of at least three experiments and shown as mean ± SD. *, P <0.05; **, P <0.01.

Journal: PLoS ONE

Article Title: Transglutaminase 2 Contributes to Apoptosis Induction in Jurkat T Cells by Modulating Ca 2+ Homeostasis via Cross-Linking RAP1GDS1

doi: 10.1371/journal.pone.0081516

Figure Lengend Snippet: (A) RAP1GDS1 and TG2 protein expression levels detected by Western blotting in Tet-on wtTG2, Tet-on wtTG2 with sh vector and Tet-on wtTG2 with shRAP1GDS1 (sh1) cells following 18 hours Dox (50 µM) treatment. β-actin was used as loading control. Tet-On wtTG2 with sh vector and sh1 cells treated with Dox for 18 hours were exposed to 5 µM thapsigargin. (B) A representative recording of the tg-induced Ca 2+ release from the ER recorded by Mag-Fura-2/AM fluorescence is shown. Right panel , statistical evaluation of the tg-induced ER Ca 2+ depletion. (C) A representative recording of the tg-induced intra-mitochondrial Ca 2+ changes is shown. Right panel , Area, statistical evaluation of integrated Ca 2+ response. AUC, area under the curve. These data are representative of at least three experiments and shown as mean ± SD. *, P <0.05; **, P <0.01.

Techniques: Expressing, Western Blot, Plasmid Preparation, Fluorescence

$H: homogenate; Mc: crude mitochondria; ER; MAM: mitochondria-associated membrane; C: cytosol. Ten µg of protein components of subcellular fractions prepared from Jurkat Tet-On cells were loaded on 10% SDS-PAGE and transferred to PVDF membrane for standard western blotting. The presence of TG2 and RAP1GDS1 were shown using specific monoclonal antibodies. Marker proteins indicate mitochondria (VDAC), ER (IP3R3), MAM (Sigma-1R) and cytosol (β-tubulin).$

Journal: PLoS ONE

Article Title: Transglutaminase 2 Contributes to Apoptosis Induction in Jurkat T Cells by Modulating Ca 2+ Homeostasis via Cross-Linking RAP1GDS1

doi: 10.1371/journal.pone.0081516

Figure Lengend Snippet: H: homogenate; Mc: crude mitochondria; ER; MAM: mitochondria-associated membrane; C: cytosol. Ten µg of protein components of subcellular fractions prepared from Jurkat Tet-On cells were loaded on 10% SDS-PAGE and transferred to PVDF membrane for standard western blotting. The presence of TG2 and RAP1GDS1 were shown using specific monoclonal antibodies. Marker proteins indicate mitochondria (VDAC), ER (IP3R3), MAM (Sigma-1R) and cytosol (β-tubulin).

Techniques: SDS Page, Western Blot, Marker

Increases in intracellular Ca 2+ concentrations trigger the crosslinking activity of TG2. TG2 crosslinks RAP1GDS1, which in turn facilitates the exchange of GDP to GTP on its target GTPase protein. This small GTPase initiates a not yet characterized signalling pathway, which enhances Ca 2+ release from the ER and the consequent Ca 2+ uptake of the mitochondria. High amounts of mitochondrial Ca 2+ sensitize for apoptosis, while physiological amounts enhance mitochondrial ATP production.

Journal: PLoS ONE

Article Title: Transglutaminase 2 Contributes to Apoptosis Induction in Jurkat T Cells by Modulating Ca 2+ Homeostasis via Cross-Linking RAP1GDS1

doi: 10.1371/journal.pone.0081516

Figure Lengend Snippet: Increases in intracellular Ca 2+ concentrations trigger the crosslinking activity of TG2. TG2 crosslinks RAP1GDS1, which in turn facilitates the exchange of GDP to GTP on its target GTPase protein. This small GTPase initiates a not yet characterized signalling pathway, which enhances Ca 2+ release from the ER and the consequent Ca 2+ uptake of the mitochondria. High amounts of mitochondrial Ca 2+ sensitize for apoptosis, while physiological amounts enhance mitochondrial ATP production.

Techniques: Activity Assay

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